We performed life-table experiments with two species of the littoral rotifers Lecane luna (O.F. Müller, 1776) and. Lecane quadridentata (Ehrenberg, 1832).
Hydrobiologia 387/388: 341–348, 1998. E. Wurdak, R. Wallace & H. Segers (eds), Rotifera VIII: A Comparative Approach. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
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Effect of temperature and food concentration in two species of littoral rotifers Ignacio Alejandro P´erez-Legaspi & Roberto Rico-Mart´ınez∗ ´ Universidad Aut´onoma de Aguascalientes, Centro B´asico, Departamento de Quimica, Avenida Universidad 940, C.P. 20100, Aguascalientes, Ags., M´exico (∗ author for correspondence) Key words: Lecanidae, life-table, mortality, reproduction rate, survivorship
Abstract We performed life-table experiments with two species of the littoral rotifers Lecane luna (O.F. Müller, 1776) and Lecane quadridentata (Ehrenberg, 1832). Three different temperatures (20, 25 and 30◦ C) and food concentrations of Nannochloris oculata (1×107, 5×106, and 1×106 cells ml−1 ) were investigated. We found important differences between both species in all the treatments regarding offspring sizes, hatching percentages, life span and reproductive rates. Our data on hatching percentages of asexual eggs suggested that the optimal temperature for both species is in the 20–25◦C range. On the other hand, reproductive data placed the optimal temperature near 25◦C. This information can be used to develop aquatic toxicology tests with littoral species.
Introduction Temperature and food are among the most important factors influencing growth and development of rotifers, which are mostly ‘r’ strategists and have a high ability to rapidly occupy new niches (Nogrady et al., 1993). There are several works in the literature that have studied the effect of environmental factors on rotifer growth and development (for a review see Nogrady et al., 1993). Galkovskaya (1987) studied the ingestion rate of Chlorella sp. cells by Brachionus calyciflorus Pallas at different food concentrations and temperatures. She found three-fold increases of the maximum ingestion rate in this species when temperature was increased from 20◦ C to 35◦C. It is wellknown that the small size and impermeability of their integument make rotifers quite susceptible to physical and chemical changes. (Nogrady et al., 1993). Rotifers produce cysts to escape adverse environmental conditions. The density of cysts and their hatching percentages in natural conditions varied among the different species investigated by May (1987). Gilbert (1995) did not observe differences in the mean number of cysts produced by Synchaeta pectinata at temperatures of 12 and 19◦C. He did find that when populations of Synchaeta pectinata were deprived of food in the
lab for long periods of time, they were able to produce cysts more frequently. The above examples clearly show that severe environmental conditions can have an important effect on rotifer populations. Therefore, it is important to develop life-table data to determine the influence of some of these factors on rotifer populations. Temperature is one of the most important factors controlling natality and growth rate in several species of rotifers (Galkovskaya, 1987; Sarma, 1985; Rao & Sarma, 1988; Sarma & Rao, 1990). Life-table experiments provide us with information about dynamic changes in populations based on observations of reproduction and mortality of independent individuals (Krebs, 1985). One of the best life-table studies of rotifers was carried out by Galkovskaya (1987). She worked with three species of rotifers; B. calyciflorus, B. urceus and Epiphanes brachionus, and found that the mean generation time was more than double the sum of the duration of embryogenesis plus the juvenile phase (De + Dj). In her experiments, the mean generation time was reduced when the temperature increased from 30 to 40◦ C, and the net reproductive rate (Ro) at 39◦C increased two-fold when food concentration increased. However, the effect of increasing food con-
342 centration was negligible at 37◦ C, and at 40◦C the net reproductive rate (Ro) was reduced significantly. Several life-table experiments have been performed so far (for a review see Nogrady et al., 1993); however, these works are mainly on planktonic species. Little information is given on littoral species. In the large genus Lecane this lack of information is an obstacle to understanding the biogeographic distribution of its numerous species. Hummon & Bevelhymer (1980) developed life-table experiments with Lecane tenuiseta using baker’s yeast as food. Their results were quite interesting in spite of the low quality food they used. Therefore, it would be quite interesting to compare their data with that obtained from life-tables where a more adequate food (such as algae) is used. Toxicity assessment is an activity that becomes more important every day. Although several rotifer species have been used to develop standard toxicity tests, most of the tests have used species of the family Brachionidae (Snell & Janssen, 1995). This may be due to their ease of culture in the lab and the advantage of ease in cysts hatching over maintenance of stock cultures (Snell & Moffat, 1992). However, these planktonic species are poor models to study sediment toxicity which can be a potential threat to rivers and water reservoirs (ReVélle & ReVélle, 1992). Therefore, the goal of our work was to determine the influence of temperature and food concentration on the two littoral species Lecane quadridentata (Ehrenberg, 1832) and Lecane luna (O. F. Müller, 1776) using life-table experiments with a more suitable food, the alga Nannochloris oculata.
Materials and methods The littoral species used in this work were collected in the field and grown in the lab for at least six months prior to experiments. Lecane quadridentata (Ehrenberg, 1832) was collected at Lake Chapala (Mexico’s biggest natural lake) and Lecane luna (O. F. Müller, 1776) was collected at Los Arquitos Dam (for location of this dam see Silva-Briano, 1992). These two species were cultured in EPA medium (U.S. EPA, 1985) and fed the green algae Nannochloris oculata (LB2194, University of Texas Collection) grown in Bold’s Basal Medium (Nichols, 1973). We studied the effect of three different temperatures (20, 25 and 30◦C), and three different food concentrations of N. oculata (1×106, 5×106 and 1×107 cells ml−1 ) in 18 combinations. The life-table started with hatching of
100 asexual eggs of each species. We observed the eggs every two hours and assigned a mean value of one hour to every individual hatched within the two hour period. Almost all animals were acclimated to the correspondent temperature for at least 48 hours prior to each experiment. There was an exception to this acclimation step. Neither species was able to initially acclimate to 30◦C and produce enough asexual eggs to allow us to produce a life table. We have data for only one treatment where acclimation was possible at 30◦ C. These data correspond to L. quadridentata fed with 5×106 cells ml−1 of N. oculata. Therefore, in this treatment we made a comparison between acclimated and non-acclimated individuals, to analyse the effect of acclimation. Hatching percentages were recorded for up to 120 hours. Neonates were then transferred to individual wells in a 24-well polystyrene plate (Corning) with the corresponding food concentration, and then incubated at the corresponding temperature in the dark. A minimum of 15 individuals were used for each treatment. Individuals were observed every twelve hours and their neonates were counted and removed from the well. Instead of changing the original individuals to new wells with fresh food (a procedure which damaged these littoral species), half of the medium was replaced every twenty-four hours by fresh medium. The algae were counted by means of a haemocytometer. The dry weight of 106 cells ml−1 of N. oculata (2.13 ± 1.90 µg for 1×106 cells ml−1 ; n=10) was determined by Standard Methods (1995) to make our results comparable with other works. The following parameters were analyzed: the twelve hours time interval (x), mean duration of lifespan (D), mean generation time (Tc), potential reproductive rate (re), net reproductive rate (Ro), and life expectancy (ex ). All of these parameters were determined according to Krebs (1985). Reproductive value (Vx ) was calculated according to Krebs (1994) and Begon et al. (1996). To obtain the mean size of individuals of both species at different ages, they were incubated at 20 ± 2◦ C with 106 cells ml−1 of N. oculata. We measured: total length, maximum width, length of the foot, and length of the pseudoclaw, according to Stemberger (1979). Morphometric data were analyzed by t-tests. Simple regression analysis between survivorship (lx) and life expectancy (ex ) for each treatment were also performed.
343 Table 1. Life-table data of Lecane quadridentata and L. luna under different temperatures and food concentrations of individually cultured cohorts
Food concentration (106 cells ml−1 )
Mean duration of lifespan (D) (h)
Mean generation time (T) (h)
Potential reproductive rate (re ) (h−1 )
1 5 10
153.7 169.0 176.1
110.0 125.6 127.7
0.0547 0.0502 0.0382
67.1 71.4 70.9
25
1 5 10
175.5 167.2 91.6
94.7 114.9 87.0
0.0753 0.0630 0.0431
145.3 127.5 27.6
30
1 5 10
88.5 45.7 32.6
64.3 73.7 0
0.0834 0.0126 0
69.2 2.4 0
1 5 10
163.3 138.5 196.5
115.9 123.8 143.5
0.0492 0.0376 0.0437
104.3 56.0 176.2
25
1 5 10
178.5 135.1 158.2
104.9 86.2 92.6
0.0771 0.0677 0.0664
232.4 118.6 141.8
30
1 5 10
85.4 62.8 51.3
60.5 45.2 0
0.0787 0.0205 0
59.9 2.5 0
Temperature (◦ C) L. luna 20
L. quadridentata 20
Results and discussion According to Galkovskaya (1987), it is of utmost importance to know the range of temperature tolerated by rotifer populations. Temperature has been shown to influence the duration of lifespan in several rotifer species (Halbach, 1970; Walz, 1983; Galkovskaya, 1987). This effect was particularly important for Keratella cochlearis (Walz, 1983), where individuals grown at 10–15◦C lived for about 47–55 days, while those raised at 20–25◦C lived for 15–20 days. We found a similar response in our life-tables for both species. At 20◦ C and the highest food concentration tested (1×107 cells ml−1 ), longevity was the highest for both species. Also, at 20◦ C increases in food concentration produced increases in longevity in both species (Table 1). In contrast, at 25 and 30◦ C when food
Net reproductive rate (Ro) (h−1 )
concentration increased, longevity decreased in both species, except for L. quadridentata at 25◦C. This result can be explained by the increases in respiratory costs due to increases in food concentration (Doohan, 1973; Rico-Martínez & Dodson, 1992). In our work, as well as in others (Walz, 1983; Galkovskaya, 1987) the effect of temperature on the mean generation time (Tc) parallels the effect on longevity. The highest values of the potential reproductive rate (re) were found at the smallest food concentration and at the highest temperature in both species, as was found by Galkovskaja although for Brachionus calyciflorus (1983), a similar value was also found at the lowest temperature and the highest concentration of food in her work. In our work, there is a positive relationship between the values of the potential reproductive rate (re) and the net reproductive rate (Ro) at 20 and
344 Table 2. Morphological characterization of individuals of different ages of Lecane luna and L. quadridentata at 25±2 ◦ C. Measurements are given in micrometers with ± one SD Age (hours)
Maximum length
Lecane luna 0 24 48 Ovigerous females∗
176.3±19.6 166.9±9.5 166.2±14.8 201.4±9.1
Lecane quadridentata 0 24 48 Ovigerous females∗
227.5±19.3 246.0±8.7 246.0±9.7 288.0±15.0
Length of the foot
Length of the pseudoclaw
Maximum width
57.0±5.5 55.0±5.6 55.7±8.2 67.7±5.5
9.6±1.2 10.6±1.1 10.1±1.0 11.1±1.3
110.2±10.3 111.0±5.9 105.9±6.4 134.5±9.1
87.6±10.3 89.6±11.0 92.7±8.4 108.6±7.3
17.4±2.2 17.1±2.2 18.6±2.7 23.1±2.3
113.1±10.6 121.0±7.3 120.7±6.2 145.2±4.0
∗ Randomly picked up from our cultures.
Table 3. Hatching percentages of two species of littoral rotifers. Hatching percentages were determined at different temperatures and times after eggs were transferred to individuals wells Species
L. luna
L. quadridentata
Temperature (◦ C)
24
Time after transfer 48 72
20 25 30 20 25 30
28.6±22.3 51.7±38.0 0 53.9±15.2 37.7±25.5 0.1±0.3
74.8±22.9 62.4±40.2 0 90.6±12.5 46.1±26.3 2.5±5.3
79.1±25.1 64.1±45.8 0 93.1±9.9 48.4±26.4 2.0±5.3
n∗
10 10 5 7 13 9
∗ Eggs were randomly collected from the cultures. n = Number of wells. Each well
contained 10 eggs.
25◦C, but there is a big discrepancy between the same parameters at 30◦ C. This discrepancy is explained by the difference in mean generation time, which drops drastically at 30◦C. The highest Ro values for both species were thus recorded at 25◦ C. From the analysis of our life-table data it is clear to us that the optimal temperature for both L. luna and L. quadridentata is probably found around 25◦ C. Galkovskaya (1983) also found similar relationships between re and Ro for B. calyciflorus, including high values of re and low values of Ro at the higher temperatures. In our life-table experiments, L. luna and L. quadridentata started reproduction after 36 hours. In one or two exceptional cases, neonates were produced at 24 hours, and at 30◦ C reproduction was delayed in some treatments or never appeared (Figure 1). We found that the reproductive value (Vx ) peaked in the lowest food concentration treatments in L. luna at 25 and 30◦ C,
Table 4. Correlation (adjusted R 2 ) between survivorship (lx) and mean life expectancy (ex) for all 18 treatments Species
Food concentration (106 cells ml−1 )
20◦ C
Temperature 25◦ C 30◦ C
L. luna
1 5 10
0.7473 0.8449 0.6492
0.7065 0.6239 0.8768 −0.0797 0.54422 0.1428
L. quadridentata
1 5 10
0.8183 0.7753 0.8470
0.7454 0.8484 0.8463
0.6881 0.8036 0.8860
345
Figure 1a. Figure 1. Influence of food concentration and temperature on the reproductive value (Vx ) of (a) L. luna and (b) L. quadridentata. The numbers: 1, 5, and 10 correspond to the food concentration in 106 cells ml−1 of N. oculata.
and at 30◦ C in L. quadridentata (Figure 1). Also, the peak occurred between 48 and 108 hours for all treatments (including 20◦ C). Our highest reproductive values are slightly higher than those of Hummon & Bevelhymer (1980) and Galkovskaya (1987). While we were developing the method to do a life table of both Lecane species, we encountered several problems. One was the injury or death of progenitors after transference to a new well with fresh medium. Therefore, we decided to remove neonates, allowing the progenitor to stay in the same well thus avoiding
the stress produced by the transfer. Another problem was the low hatching percentage of asexual eggs at 30◦ C (Table 3). For that reason we were unable to perform the life-table experiments at 30◦ C under the same pre-conditions as those at 20 and 25◦ C. To assess the effect of acclimation to the previous generation (Galkovskaya, 1983) we performed an additional lifetable experiment using animals obtained from hatching asexual eggs at 30◦ C (produced by individuals previously acclimated at 30◦ C for at least 48 hours). We compared survivorship (lx) and fecundity (mx) for
346
Figure 1b. For legend see p. 345.
acclimated vs. non-acclimated animals (Figure 2). Acclimated animals live longer and are more fecund. A similar result was reported by Galkovskaya (1983), who reported considerable mortality of animals at higher temperatures during acclimation. Similarly, in our experiments, after keeping the cultures of both species for up to two weeks at 30◦C, all animals died. In contrast, cultures at 20 and 25◦ C have been kept for years in our lab. This observation confirms the inadequacy of 30◦ C for culturing these species. Perhaps the ability to tolerate high temperatures for up to two weeks confers on these two species an ecological advantage in occupying temporally a particular niche.
Galkovskaya (1983) discussed the probability of using ‘thermal selection’ for producing highly efficient rotifer cultures, even when parthenogenesis is the only mechanism of reproduction. Hummon & Bevelhymer (1980; Figure 1) found a strong relationship between survivorship (lx) and life expectancy (ex ) for Lecane tenuiseta. We investigated that same relationship for L. luna and L. quadridentata in our work, and in general we found high correlations between these two parameters (Table 4). The only two exceptions were in the two highest food concentration treatments for L. luna at 30◦C. In these two cases correlations are lower than 0.50.
347
Figure 2. Comparison of acclimated vs. non-acclimated L. quadridentata individuals. The data correspond to the 5×106 cells ml−1 treatment at 30◦ C. (a) Survivorship (lx) comparison. (b) Fecundity (mx) comparison.
We performed two individual tailed t-tests to analyze the morphometric data in Table 2. As a result of that analysis, it is clear that L. quadridentata grows little from 24 to 48 hours. A comparison of maximum length (ML) and maximum width (MW) for L. quadridentata showed no significant differences between these two age groups (p=0.99 in both cases). However, significant differences were found between neonates and either 24- or 48-hour-old animals and between ovigerous females (adults) and all other groups (p
